The formation of gastric ulcer was assessed pathologically using both ulcer index (UI) and ulcer score. The whole stomach tissue was harvested, opened along the greater curvature, and the mucosa was exposed for ulcer evaluation. The opened stomach tissue was fixed on the cork board with fine needles, and red coloration and hemorrhagic streaks in the ulcerated area were observed. The ulcerated area was assessed using planimetry under 1 mm × 1 mm graph paper with recording camera. Ulcer index was calculated using ImageJ software as below [25]:

The excised stomach tissue (5 mm × 5 mm) from the normal or lesion area was preserved in 10% buffered formalin overnight, embedded in paraffin wax, and stained with H&E to observe the damaged stomach. Coded specimens were evaluated by a pathologist in a blinded fashion. Ulcer score was evaluated under a light microscope according to the following criteria: 0: normal coloration, 0.5: red coloration, 1: spot ulcers, 1.5: hemorrhagic streaks, 2: ulcers > 3 mm but < 5 mm, and 3: perforation [25].

The development of gastric ulcer was monitored biochemically using serum hydroxyproline levels. Hydroxyproline has been considered as a serum biomarker for gastric ulcer in rats [26]. A decrease in serum hydroxyproline levels reflected the development of gastric ulcer by aspirin, alcohol, or stress in rats [26]. Serum hydroxyproline were assessed by an enzymatic colorimetric method using a commercial reagent kit (CSB-E08838r, Cusabio Biotech Co., Ltd., College Park, MD, USA), and the absorbance was measured at 450 nm and corrected at 540 nm.

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